Gene therapy vehicle comprising dermal sheath tissue

ABSTRACT

The invention herein described relates to a gene therapy vehicle, comprising dermal sheath tissue and/or cells derived from portions of hair follicles which show pluripotentiality, and which has use in the delivery of therapeutic agents to selected tissues and advantageously has the potential to repair/replace damaged tissue.

The invention relates to the use of dermal sheath tissue and/or cellsderived therefrom and/or portions of hair follicles containing these andother cell populations for use particularly, but not exclusively in genetherapy/vaccine development.

Human gene therapy vectors constructed to date are typically derivedfrom viruses (1). The rationale being that such vectors can easilypenetrate cells by virtue of naturally infecting human cells and so canincorporate fragments of foreign DNA into a target cell population. Themost widely investigated viruses are of the adenovirus, retrovirus,parvovirus and herpesvirus families. With the exception of retroviruses,all have been derived from viruses originally isolated from humans. Innearly every case the vectors used in both ex and in vivo work have beenderived from virus mutants originally created to study gene function,rather than to act as gene delivery systems.

Although adenoviruses have proved to be popular because of ease ofgrowth of stocks to high titre, they have many associated problems. Forexample it is known that viruses which are replication incompetent incell culture have caused tissue damage and respiratory disease inpatients treated with such vectors (2).

Herpesvirus vector development to date has concentrated on derivativesof the common human pathogen herpes simplex virus (HSV). The advantageof using this virus is that it is the most intensively studied of allthe herpesviruses. The sequence of the virus genome has been determined,there is a wide range of well characterised virus mutants available andtranscriptional control processes are well understood. However thedisadvantage with this virus is that the mutant virus is difficult toproduce as high titre stocks and in some cases has an unacceptablereversion frequency. Additionally, it is a likely problem with HSVvectors that there is an innate immune response present in the majorityof the population; it is predicted that HSV vectors will suffer the sameproblem as those derived from human adenoviruses, when delivered to animmunologically competent site.

Additionally, and more recently, naturally occurring specific cellpopulations have been investigated as gene therapy delivery systemshowever such systems have to date only employed self-derived cells andconsequently are limited to the disease state of the individual fromwhich the cells are derived. Such systems suffer from immunologicalrepercussions and have not produced particularly encouraging results nordo they offer the possibility of inter and/or intra species therapies.

Thus a vehicle capable of efficient and immunopriviledged gene deliveryto human cells would have a wide range of uses in human gene therapy,for example delivery of a correct copy of human tumour suppressor genesto tumours of a variety of different organs and/or as a vaccine deliveryvehicle to induce specific immunity.

Skin is a highly complex organ covering the external surface of the bodyand merging, at various body openings, with the mucous membranes of thealimentary and other canals. It has multiple functions such aspreventing water loss from the body, but predominantly acts as aprotective barrier against the action of physical, chemical andbacterial agents on deeper tissues. Skin is elastic and except for a fewareas such as the palms, soles and ears it is loosely attached tounderlying tissue. It varies in thickness from 0.5 mm (0.02 inches) onthe eyelids to 4 mm (0.17 inches) or more on the palms and soles.

Skin is composed of two layers (please refer to FIG. 1 which illustratesan anatomical cross-sectional view through a slice of skin), the outerlayer, which is comparatively thin (0.1 mm) is called the epidermis, orcuticle, it is several cells thick and has an external, horny layer ofdead cells that are constantly shed from the surface and replaced frombelow by a basal layer of cells, called the stratum germinativum. Theepidermis is composed predominantly of keratinocytes which make up over95% of the cell population, the rest include dendritic cells such asLangerhans cells and melanocytes. It is essentially cellular andnon-vascular, there being relatively little extracellular matrix exceptfor the layer of collagen and other proteins beneath the basal layer ofkeratinocytes. Keratinocytes of the basal layer are constantly dividing,and daughter cells subsequently move outwards, where they undergo aperiod of differentiation and are eventually sloughed off from thesurface. The inner layer of the skin is called the dermis and iscomposed of a network of collagenous extracellular material, elasticfibres, blood vessels and nerves. Contained within it are hair follicleswith associated sebaceous glands (collectively known as thepilosebaceous unit) and sweat glands. The interface between theepidermis and dermis is extremely irregular and consists of a successionof interdigitations, or finger like projections. Beneath the basalepidermal cells along this interface the specialised extracellularmatrix is organised into a distinct structure called the basementmembrane.

The mammalian hair fibre is the product of a small but complex,cylindrical arrangement of tissues known as the hair follicle. Follicleslie angularly underneath the skin's surface, their distal most epidermisbeing in direct continuation with that of the skin at the point wherethey open externally. Although small, the follicle comprises a highlyorganised system of recognisably different layers arranged in concentricseries. Active hair follicles extend down through the dermis, thehypodermis (a loose layer of connective tissue), and the fat or adiposelayer.

At the base of any active follicle lies the hair bulb, which consists ofa body of dermal cells, known as the dermal papilla, contained in aninverted cup of epidermal cells known as the epidermal matrix (pleaserefer to FIG. 1). Irrespective of follicle type, the hair fibre,together with several supportive epidermal layers, is produced bygerminative epidermal cells at the very base of this epidermal matrix.The lowermost dermal sheath is contiguous with the papilla basal stalk,from where it curves externally around all of the epidermal layers ofhair matrix as a thin covering of tissue, and then continues as a tubeor sleeve for the length of the follicle. The dermal sheath is otherwiseknown as the connective tissue sheath.

Developing skin appendages such as feather and hair follicles rely oninteraction between the skin's two layers, the epidermis and the dermis.In embryonic development, a sequential exchange of information betweenthese layers underpins a complex series of morphogenetic processesculminating in the formation of adult follicle structures. However,following maturity, and in contrast to general skin dermal and epidermalcells, certain hair follicle cell populations retain embryonic-typeinductive, interactive and biosynthetic behaviours. These properties arelikely to derive from the very dynamic nature of the cyclicallyproductive follicle, in which repeated tissue remodelling necessitates ahigh level of dermal-epidermal interactive communication, as is vitalfor embryonic development and, as would be desirable in any form oftissue reconstruction.

Hair fibre is produced at the base of an active follicle at a very rapidrate (0.4 mm per day in the human scalp follicles and up to 1.5 mm perday in the rat vibrissa or whiskers), which means that cellproliferation in the follicle epidermis ranks amongst the fastest inadult tissues (3).

The most dynamic region of the hair follicle is the deeply embedded endbulb, where local dermal-epidermal interactions drive active fibregrowth. This same region is also central to the developmental changesand tissue remodelling involved in the hair fibre's or appendagesprecise alternation between growth and regression phases. As a keyplayer in the activities, the dermal papilla appears to orchestrate thecomplex program of differentiation that characterises hair fibreformation from the primative germinative epidermal cell source (4-7).The lowermost dermal sheath initiates below the papilla basal stalk,from where it curves outwards and upwards to externally enclose all ofthe layers of the epidermal hair matrix as a thin cup of tissue. (Pleaserefer to FIG. 1). The dermal sheath continues as a tubular arrangementfor the length of the follicle, as does the epidermal outer root sheathwhich lies immediately internal to it in between the two layers is aspecialised basement membrane termed the glassy membrane. The outer rootsheath constitutes little more than an epidermal monolayer in the lowerfollicle, but becomes increasingly thickened more superficially.

Whilst the individual anatomical components and cell sub-populations ofskin are well established their intra/inter biochemical interactions andcontrol mechanism remains largely a matter for speculation and intenseresearch.

The most important of all cells types are those at the source of everybiological system ie stem cells, since they vitally sustain andreplenish the more differentiated descendent population and as theybecome specialised develop a characteristic function. Yet these are thecells which are least understood in terms of their distribution,behaviour and the actors by which they may be defined. The ability toprovide significant numbers of pure, unstimulated, undifferentiated,primitive stem cells from an adult organ would be likely to have a broadimpact on our fundamental understanding of cell biology, and would yieldpositive and promising approaches to future therapeutic advances.

Serendipitously, we have studied hair follicles and identified aspecific cell population with immunoprivilege and stem specific cellpotential that can be used most advantageously as a cellular deliverysystem in gene therapy.

We have found that the implantation of male follicle-derived dermalsheath cells into a female recipient does not lead to the typical immuneresponse and subsequent rejection that one would expect. The sameobservation held true even after a subsequent set of implantations withthe same human host and donor, when second set rejection would have beenpredicted. Such results show that dermal sheath cells have some form ofprivileged immune status. A number of our tissue interaction/inductionstudies have also clearly demonstrated that cells derived from differentspecies appendages are very capable of interacting with each other, andcommunicating at the appropriate levels to allow complex morphogenesis.This being the case, dermal sheath tissue and/or cells derived therefromrepresent a cell population of major consequence in gene therapy asvehicles for both inter and intra species therapy delivery. Additionallythe ability of dermal sheath tissue and/or cells derived therefrom todifferentiate into a variety of different phenotypes makes theircontribution to gene therapy even more significant, in that using suchcells as vehicles means that not only would they be tolerated inmultiple and different tissue/cell sites, but that they would also bemore effective and penetrating by differentiating into multiple tissuetypes depending on the site of delivery. Further natural attributes thatpre-dispose follicle cells as candidates for the application of genetherapy include: their similarity to wound myofibroblasts; theirexhibition of stem cell-type qualities including those characterisingprimative muscle lineages, indeed follicle-derived muscle stem cells areespecially suited to gene therapy applications because of their abilityto fuse with other cells. Furthermore since dermal sheath cells havemany of the properties of smooth muscle cells they have an additionalpotential in vascular related therapy by incorporation into bloodvessels as the smooth muscle component; production of a uniqueembryonic-type extracellular matrix and, the fact that they exhibitimpressive regenerative and inductive abilities.

It is therefore an object of the invention to provide a new gene therapysystem that employs follicle derived cells/tissues and/or theirattributes.

It is a yet further object of the invention to provide an inter or intraspecies gene therapy that employs follicle derived cells/tissues and/ortheir attributes.

It is a yet further object of the invention to provide a gene therapyvehicle having multi-potential incorporation and differentiationproperties.

According to a first aspect of the invention there is provided dermalsheath tissue and/or cells derived therefrom and/or cells typicallyclosely associated with hair follicles for use in a gene therapy.

According to a further aspect of the invention there is provided a genetherapy vehicle for delivering at least one selected gene, or functionalfragment thereof to a target site comprising dermal sheath tissue and/orcells derived therefrom and/or cells typically closely associated withhair follicles.

Reference herein to a functional fragment thereof is intended to includea part of a gene that provides for the expression of the correspondingprotein or an active or effective part thereof.

Reference herein to cells typically closely associated with hairfollicles is intended to include cells that are functionally and/orlocationally associated with and/or within hair follicles.

In a preferred embodiment of the invention said dermal sheath tissueand/or said cells derived therefrom and/or said cells typically closelyassociated with hair follicles is/are derived from a selected portion ofa follicle ideally the lower third thereof and even more ideally arederived from a segment or ring of a combination of follicletissue/cells.

In a yet further preferred embodiment of the invention said gene therapyvehicle is suitably engineered, ideally using recombinant techniques, soas to include at least one insertion site into which at least oneselected gene can be placed. Those skilled in the art will appreciatethat the provision of this insertion site allows the gene therapyvehicle to carry a selected gene to a desired location. More preferablystill said selected gene is functionally inserted into said gene therapyvehicle so that the expression of said gene results in the provision ofthe corresponding protein product. It would be understood by thoseskilled in the art that the nature of the gene to be inserted will beselected having regard to the purpose of the gene therapy vehicle andthus the nature of the condition to be cured, treated or alleviated. Inaddition, said gene therapy vehicle may be provided with multipleinsertion sites with a view to carrying multiple genes and so providingfor the delivery of multiple proteins, either of a similar or differentnature. In each instance, said selected gene for insertion is arrangedso as to be inserted in-frame with the genome of the gene therapyvehicle so as to provide for correct expression of same.

In a yet further preferred embodiment of the invention said gene therapyvehicle comprises at least one selected gene or functional fragmentthereof which is operationally attached to a regulatable or inducible ora constitutive promoter.

In a yet further aspect of the invention there is provided a vector fortransforming or transfecting the gene therapy vehicle of the inventionwherein said vector is provided with at least one insertion site intowhich at least one selected gene can be placed and also other expressioncontrol elements for ensuing that once the vector infects or penetratessaid tissue and/or cells derived therefrom expression of the saidselected gene can take place.

In a yet further preferred embodiment of the invention there is provideda therapeutic composition comprising a suitable carrier for the genetherapy vehicle in accordance with the invention, ideally said carriercan be formulated to have anti-bacterial properties and/or anti-septicproperties and more ideally further include growth promoting additivesand/or local anaesthetics. Ideally said therapeutic composition may beadapted to be applied topically in the form of dermal sheath cellssuspended in a suitable carrier solution/gel/cream/emollient;alternatively said composition may be adapted to be administered byinjection and so comprise a carrier solution; alternatively still, saidcarrier may be incorporated and/or embedded therein and/or associatedtherewith and/or attached thereto a plaster or bandage or the like.

According to a further aspect of the invention there is provided apotential gene therapy vehicle for use in delivering a selected gene, orfunctional fragment thereof, to a given site wherein said gene therapyvehicle comprises dermal sheath tissue and/or cells derived therefromand/or cells typically closely associated with hair follicles whichcells and/or tissue that have been suitably adapted to accommodateheterologous genetic material and which, in vivo, have the capacity toselectively differentiate to provide at least one differentiated tissuetype.

It will be apparent to those skilled in the art that, given thepluripotentiality of these cells, that the site of implantation will, tosome extent, determine the differentiated pathway along which thesecells will develop. Thus, the site of implantation will determine thenature of the phenotype of these cells and therefore one is providedwith a gene therapy vehicle that not only is able to deliver at leastone selected gene but which also has the added advantage of being ableto provide differentiated tissue. This feature is particularly importantwhere an individual may have suffered tissue damage, for example,following wounding of any type or following ischemia or vascular damage,or even removal of at least part of an organ or tissue.

It would therefore be seen that, advantageously, the gene therapyvehicle of the invention may be suitably cultured for the purpose ofimplantation and/or suitably impregnated onto wound healing materialssuch as bandages or seeded into biomaterials or coated onto replacementblood vessels or the like.

In the instance where the gene therapy vehicle is to be used in relationto wound healing said dermal sheath tissue and/or said cells derivedtherefrom and/or cells typically closely associated with hair folliclesare provided or combined with at least one other appropriate cell typefrom a hair follicle. This combination is favoured because ourexperiments have shown that dermal papilla tissue, or cells derivedtherefrom may assist in the closure of wound and in the reduction ofscar tissue.

In a yet further preferred embodiment of the invention there is provideda wound healing system comprising a suitable matrix material havingincorporated and/or embedded therein and/or associated therewith and/orattached thereto a gene therapy vehicle in accordance with theinvention, ideally said matrix material comprises native collagen orcollagenous gels or lattices constructed from reconstituted collagen orhighly complex mixtures of reconstructed collagen and a multitude ofextracellular matrix products or any other suitable matrix materialknown to those skilled in the art, the selection of which is notintended to limit the scope of the invention.

In a yet further preferred embodiment of the invention there is provideda surgical dressing comprising a web material and a suitable matrixmaterial, at least one of which materials has incorporated and/orembedded therein and/or associated therewith and/or attached thereto agene therapy vehicle in accordance with the invention, ideally saidsurgical dressing is conventional, the selection of which is notintended to limit the scope of the invention.

According to a yet further aspect of the invention there is provided awound healing system as hereinbefore described for use in treatment ofacute and/or chronic and/or minor and/or severe wound healing; and/orcartilage repair and/or bone repair and/or muscle repair and/orconnective tissue repair and/or blood vessel repair.

In summary, we believe the dermal sheath tissue and/or cells derivedtherefrom and/or cells typically closely associated with hair follicleshave an important part to play in gene therapy because this tissueand/or cells derived therefrom and/or cells typically closely associatedwith hair follicles:

-   i) exhibit immunoprivilege,-   ii) exhibit the capacity to incorporate themselves within disturbed    tissue sites and fuse directly with host cells,-   iii) exhibit multipotentiality in terms of the differentiated cell    lineages they can follow,-   iv) exhibit interactive flexibility both in terms of merging within    different body sites and also surviving and interacting within    different species,-   v) exhibit longevity and general durability, e.g. can be stored long    term at low temperatures and still retain the aforementioned    properties,-   vi) advantageously are of adult origin and since most gene therapies    will be aimed at adults the gene therapy of the invention provides    the benefits of embryonic-type properties without the potential risk    of utilising genuine embryo derived cells,-   vii) represent a relatively rich deposit of stem cells,-   viii) promote healing thereby reducing scarring and delay    fibro-fatty deposit accumulation,-   ix) have the ability to pass through the basement membrane, by    virtue of the production of large amounts of metalloproteinases, as    seen in lower follicle regeneration when sheath cells move through    basement membranes on the way to becoming papilla cells (8, 9), thus    these cells have the potential to reach parts of the body remote    from site of delivery.

Thus the invention presents a gene therapy delivery system that can bereliably manufactured and then stored for future use. Additionally, thistissue and/or cells derived therefrom can exist for a long time inculture under extreme stress, and accordingly presents a gene therapydelivery system that is robust in nature, another favourable advantagein terms of storage, and subsequent application.

An embodiment of the invention will now be described by way of exampleonly with reference to the following Figures wherein:

FIG. 1 represents a diagrammatic illustration of an anatomicalcross-sectional view through a slide of skin:

-   A external hair fibre;-   B interfollicular epidermis of skin;-   C general interfollicular dermis;-   D sebaceous glad;-   E epidermal outer root sheath (shown in solid black);-   F dermal sheath (broken line l);-   G epidermal inner root sheath (thin layer around fibre);-   H dermal papilla (central pear shape);-   I germinative epidermal cells (form a tight collar around papillar    base).

FIG. 2 represents a diagrammatic representation of procedures;

-   A. male scalp-   A1 heals and upper follicle portions regenerate to restore    pre-biopsy state-   B. punch biopsy taken-   B1. Punch biopsy replaced on scalp-   C. end bulbs amputated-   D. end bulbs dissected-   E. to provide various tissue components-   F. isolated papilla-   G. isolated sheath-   H. pooled dermal papillae-   I. pooled dermal sheath-   J. sheath and papillae transplanted into female forearm skin-   K. female arm where male tissue has induced hair follicle    neogenesis!

FIG. 3 represents pictorial evidence of isolated dermal papilla (P) andsheath (S) tissue microdissected from male scalp hair follicle endbulbs: as shown in FIG. 2 e, marked by a star (*).

FIG. 4 represents pictorial evidence of two hair fibres which have beenproduced in the immediate vicinity of the male dermal sheath-implantedfemale skin wound protected by a small silicone rubber collar.

FIG. 5 represents pictorial evidence of FIG. 4 after the silicone collar(and plaster attachment) has been removed.

FIG. 6 represents pictorial evidence of a histological section throughan end bulb region of an induced follicle, revealing an Alcianblue-positive stained papilla (P).

FIG. 7 represents pictorial evidence of a lower portion of an inducedfollicle which can be seen to stain positively following in situhybridisation with a Y-chromosome-specific DNA probe, realised viadigoxygenin label.

FIG. 8 represents pictorial evidence of a tissue section acting asnegative control for FIG. 7 and represents female skin that is notstained at all by the digoxygenin-linked Y-chromosome probe.

FIG. 9 represents pictorial evidence of a lower portion of an inducedfollicle stained positively following in situ hybridisation with aY-chromosome-specific DNA probe, realised via a green fluorophoremarker.

FIG. 10 represents pictorial evidence of a tissue section acting as apositive control for FIG. 9.

FIG. 11 represents pictorial evidence of a high power magnification viewof the side of a long term [24 days] graft.

FIG. 12 represents pictorial evidence of dermal sheath cell capabilityto differentiate into different mesenchymal cells.

-   (A) Long tern cultured (over a year) human dermal sheath cells-   (B) Dermal sheath cells appearing to fuse in myoblast (muscle-lil-   (C) Myotube-like structures in dermal sheath cell cultures.-   (D) Adipocyte (fit producing) cells.-   (E) Chondrocyte (cartilage-type) cells.-   (F) Mineral producing bone precursor cells—Von Kossa stained.-   (G) Dermal sheath cells labelled immunohistochemically for    alpha-smooth muscle actin.-   (H) Human dermal sheath cells positively stained for smooth muscle    myosin.-   (I) Dermal sheath cells labelled positively for desmin.

FIG. 13 represents pictorial evidence of skin at the margin of a woundand in which dermal sheath cells have surrounded an isolated follicle inthe undamaged tissue away from the main group of labelled cells remotein undamaged tissue.

FIG. 14 represents a schematic representation of an e-GFP construct fortransforming dermal sheath cells.

FIG. 15 represents a schematic representation of a method for insertingthe e-GFP gene into a vector.

FIG. 16 represents pictorial evidence of transfected dermal sheath cellswith a construct containing enhanced green fluorescent protein e-GFP anda constitutive promoter.

EXPERIMENTAL APPROACH

Tissue Isolation

A small patch of male scalp skin (about 1.5 cm²) was coarsely shaven,leaving some fibre still exposed to allow for subsequent plucking. Thearea was wiped with an antiseptic solution and injected locally withlignocaine plus adrenaline anaesthesia, before taking a 6 mm diameterpunch biopsy at an angle appropriate to follicle orientation. The mostproximal tips (under ⅕th of length) of the exposed follicles wereamputated under a dissecting microscope (Zeiss) from the invertedbiopsy, and transferred to individual drops of minimal essential medium(Gibco) at 4° C. After plucking the hair fibres from the transectedfollicles, the biopsy was returned to its original scalp skin site andleft to heal. This initial procedure lasted about 20-25 mins. Refer toFIG. 2 (a, a1, b, b1 and c) which represents a diagrammaticrepresentation of procedures.

The outermost end bulb dermal layers were inverted to allow theepidermal matrix (including undifferentiated issue) to be scraped awayand discarded (FIG. 2 d). Dermal papillae, isolated by basal stalkseverance (FIG. 2 e), were pooled in fresh medium (FIG. 2 h). The thinexternal covering of connective tissue was then teased from the piecesof sheath dermis before they were similarly pooled in fresh medium.(FIGS. 2 g and i). FIG. 3 represents pictorial evidence of isolateddermal papilla (P) and sheath (S) tissue microdissected from male scalphair follicle end bulbs as shown in FIG. 2 e marked by a star (*).

Implantations

These operations were so minimally invasive as to be practicallyimperceivable, hence, no form of local anaesthetic pretreatment wasdeemed necessary. This also avoided the possibility that the anaestheticmight adversely affect the tiny quantities of vulnerable dermis thatwere to be implanted.

A small, shallow wound was made in the inner forearm of the femalerecipient with the point of a scalpel blade, and; widened slightly usingthe tips of very fine (No.5) watchmakers forceps (FIG. 2 j). In the fewinstances when a tiny amount of blood or fluid was exuded, it wasabsorbed using tiny sterile cotton wool balls. Two sets of operationswere performed.

In the first, dermal sheath tissue from twelve follicles were implantedinto two wound sites (six in each), approximately 10 hours after the endbulbs had been removed from the biopsy. The second, involved theimplantation of 11 pieces of dermal sheath into one wound site, 9 dermalpapillae into a second, and 2 papillae (which stuck to the forceps andhad to be re-implanted separately) into a third, about 20 hours afterbiopsy. In all cases, the material was collected in as little fluid aspossible and then transferred directly to the wound site, so that itcould be rapidly inserted into the skin on the end of the forceps. Thewounds were initially left untreated and uncovered. When hair fibreswere seen emerging from the implanted sites (3-4 weeks later), smallsilicone rings with rims were placed over them and secured usingsurgical tape—as a cautionary measure to protect against abrasion,please refer to FIG. 4 which represents pictorial evidence of two hairfibres which have been produced in the immediate vicinity of the maledermal sheath-implanted female skin wound protected by a small siliconerubber collar and FIG. 5 which represents pictorial evidence of FIG. 4after the silicon collar (and plaster attachment) have been removed.

The first set of two wound sites were biopsied together as a singlepiece of elliptical skin, 77 days after sheath tissue implantation, andwere fixed immediately in freshly prepared 4% paraformaldehyde at pH7.3. The second set of wounds (made 3 months after the first) weretreated similarly—being removed 42 days post operatively as two small (4mm) punch biopsies (more precisely located by their positioning next tomoles). Detailed external observations and photographic recordings ofthe male donor scalp, and recipient female arm skin sites, were made atregular intervals.

Fluorophore-Labelled Y-Chromosome Probe [Imagenetics]

The spectrum green fluorophore-labelled enumerator probe (Imagenetics),consisted of chromosome-specific sequences from highly repeated humansatellite DNAs. The target DNA in the tissue sections was denatured in70% formamide/2×SSC at 70° C. for 10 mins. Meanwhile, the probe mixturewas prepared to contain: 7 μl SpectrumCEP hybridisation buffer (dextransulphate, formamide, SSC, pH 7.0), 1 μl SpectrumCEP probe(fluorophore-labelled enumerator probe and blocking DNA in Tris-EDTAbuffer) and 2 μl of 5× blocking solution (× number of slides), whichwere centrifuged (1-secs), heated for 5 mins in a 75° C. water bath andthen placed on ice. The denatured slides were washed in 70%, 85% and100% ethanol (1 min in each) and then air dried. Each slide, heated to45° C., received 10 μl of probe mix and then a silanised coverslip whichwas sealed at the edges prior to the slides incubation in a humid box at42° C. for 18 hours. Following hybridisation and coverslip removal, theslides were washed for: 3×10 mins in 50% formamide/2×SSC; 10 mins in2×SSC, and 5 mins in 2×SSC/0.1% NP-40, all containing Denhardtssolution, 50 μg/ml sonicated salmon sperm DNA, 1% milk powder and 0.1%Tween-20 and all at 45° C. The slides were allowed to air dry in thedark, and then 10 μl of propidium iodide counterstain (Imagenetics) anda coverslip, added to each

Digoxigenin-Labelled Y-Chromosome Probe [Boehringer Mannheim]

Each slide received 20 μl of the hybridisation mixture, consisting of:10 μl formamide [50% of final volume]; 5 μl 4× hybridisation solution;2.5 μl probe [50 ng]; 2.5 μl 8× blocking solutions. The mixture wascovered by a silanised glass coverslip, sealed and then denatured for5->10 mins at 72° C. on a pre-warmed glass plate in the oven, beforeincubation in a moist chamber at 37° C. overnight. The slides werewashed for 3×5 mins in 2×SSC, prior to 30 mins in 50 ml TBS containing1× blocking solution (as above) and 1% Boehringer kit blockerreagent—both also at 37° C. To promote detection, the slides weretransferred to 50 ml TBS and 50 μl anti-digoxigenin alkaline phosphataseconjugate [200 μg/ml] containing 1% kit blocker reagent for 30 mins at37° C., and then they were washed for 3×10 mins in 0.2% Tween 20 in TBSat room temperature. Immediately before use, 4.5 μl of NBT, 3.5 μl ofX-phosphate and 0.24 mg of levamisole (Sigma) was added to 1 ml ofTris/NaCl/MgCl₂ buffer. Appropriate volumes for the number and size ofthe sections were added and the slides incubated at room temperature ina humidified box covered in foil until the dark blue/purple colourdeveloped. To stop the reaction, the slides were rinsed for 5 mins atroom temperature in 10 mM Tris-Cl/1 mM Na2 EDTA, pH 8.0.

Sections to be counter stained with propidium iodide were incubated for5 mins at room temperature in the dark in 50 ml TBS+5 ul propidiumiodide [1 mg/ml], or a similar concentration of acriflavine yellow,washed for 2-3 mins under running water, and then allowed to air dry inthe dark. Finally, the sections were mounted in 20 μl of anti-fadingsolution under a glass coverslip, which was sealed at the edges withnail varnish

Transfection of Dermal Sheath Cells Cultured from Rat Vibrissa Follicles

Rat dermal sheath cells cultured from vibrissa follicles weretransfected using lipofectamine, according to the following procedure.1-3×10⁵ cells were seeded per well in 2 ml of the appropriate completegrowth media and plated into a six-well or 35-mm tissue culture plate.The cells were then incubated at 37° C. in a CO₂ incubator until thecells were 50-80% confluent. This procedure usually lasted 18 to 24hours. The following solutions were prepared for each transfection,solution A contained dilute 1-2 g of DNA into 100 μg serum free medium,typically OPTI-MEM® reduced serum medium (GIBCO BRL CAT.NO.31985).Solution B contained for each transfection, dilute 2-25 μl oflipofectamine reagent into 100 μl of serum free medium. Subsequently thesolutions A and B were mixed gently and incubated at room temperaturefor 15 to 45 minutes so as to allow the DNA liposome complexes to form.Further serum-free medium was added to each tube containing thecomplexes, and cells were incubated with complexes for 2 to 24 hours at37° C. in a CO₂ incubator. Following incubation, 1 ml of growth mediumcontaining twice the normal concentration of serum was added withoutremoving the transfection mixture. The medium was replaced with freshcomplete medium at 18 to 24 hours following start of transfection. Cellswere active for gene activity 24 to 42 hours after the start oftransfection.

Insertion of eGFP Gene into the Vector

The eGFP gene was cut out of the Clontech vector (GenBank Accessionnumber U55761, Catalog number 6086-1) using Hind III and Not I at themultiple binding site region (FIG. 15). The eGFP gene was then clonedinto the Invitrogen vector (pc DNA1/Amp; 4.8 kb) at the site just afterthe P cmv consitutative promoter using Hind III and Not I in accordancewith the method as outlined in FIG. 15, so that the final construct isas per represented in FIG. 14.

Storage of Dermal Sheath Tissue

Cold temperature storage of dermal sheath tissue/cells; additionallytheir subjection to adverse conditions to highlight stem cell-typecharacteristics—including capacity for preferential survival.

Human skin samples (as detailed directly above) were cleaned andappropriately microdissected to provide: (a) 3 mm² portions of wholeskin; (b) isolated hair follicles; (c) fragments of glassy membranesandwiched between thin layers of sheath dermis and ORS epidermis, and(d) primary cultures of dermal sheath cells (prepared as above). Each ofthese four levels of tissue complexity were then subjected to sixdifferent forms of adverse conditions (each repeated with and withoutserum, and/or, glucose and glycerol): (i) prolonged cold temperatestorage at 4° C.; (ii) repeated freeze/thaw cycles at −20° C.; (iii)repeated freeze/thaw storage at −80° C. in DMSO;

Results

Sheath Implants

All of the sites that had been implanted with dermal sheath tissuehealed rapidly and in a manner that seemed typical of any superficialskin lesion. Fine narrow scabs formed as the site dried and then werelost over the next few days to leave a very flint wound, which wasalmost imperceivable by about the 10th day. There was no external signof any inflammatory reaction in or around the wounds, nor any physicalperception of the site. The tip of a fibre that was darker anddisproportionately sturdier for its length than any of the arm skinslocal vellus hairs, was first noticed on the 24th day after the dermalsheath had been introduced. On the 33rd day post-implantation, a secondmuch finer and unpigmented fibre was seen to have emerged just to theside of the first. A very light peppering of pigmented material was alsovisible below the surface of the skin, in the immediate vicinity of thehealed sites. In addition, a dark line of material could be seenunderneath the skin directly behind the base of the larger fibre (referto FIGS. 4 and 5). This almost certainly represented a continuation ofthe exposed length of hair, and indicated that the follicle producing itwas shallowly embedded and at an unusual angle and orientation relativeto the local follicles. Both fibres increased in mass and length overthe next few weeks, but this was more pronounced in the pigmented fibrewhich became more obviously stouter and thus morphologically distinctfrom the local hairs (refer to FIGS. 4 and 5). The finer white fibre wascovered by a thin layer (or sac) of dried cells, but otherwise, wasquite similar in stature and general appearance to the neighbouringnon-induced hairs. Twenty one days after the second set of operations(initiated three months after the first) a fibre (again darker andsturdier than the local hairs) was seen at the sheath-implanted site.Over a further similar time span of three weeks, this solidly pigmentedhair grew thicker and became more curved. The site was biopsied on day42.

Histological examination of the sheath-implanted sites confirmed thatthe two larger follicles which had produced terminal-type fibresexternally, had all of the characteristic components. For instance,large oval (Alcian blue-positive) dermal papillae (FIG. 6, legend P)were overlaid by a pigmented epidermal matrix, and follicle-typicalconcentric tissue layers could also be clearly seen in transversesections. However, these follicles were quite different from the localvellus population in terms of their: larger size; shallow depth ofgrowth within the skin, and unusual angle of orientation parallel to theskin surface. Such independent and contrasting features strongly suggestthat the larger appendages were induced.

Notably, none of the transplanted material was transplanted into animmunoprotected site.

Further smaller follicles were also noted in random positions andarrangements in and around the post-experimental wound sites, and whilethey too may have been newly formed, their situation could not beinterpreted on the basis of the morphological criteria alone.

Evidence in Support of Immunoprivilege as Illustrated by In SituHybridisation

Both positive (refer to FIG. 7 which represents pictorial evidence of alower portion of an induced follicle which can be seen to stainpositively following in-situ hybridisation with a Y-chromosome-specificDNA probe, realised via digoxygenin label) and negative (refer to FIG. 8which represents pictorial evidence of a tissue section acting as anegative control for FIG. 7, and represents female skin that is notstained at all by the digoxygenin-linked Y-chromosome probe) controlsstained appropriately to confirm the validity of the protocols basicmethodology.

In the first set of experimental tissue sections, both of theY-chromosome-specific DNA probes recognised some of the smallerfollicles in the wound sites, as well as the more predictably inducedlarger ones. Only the lowermost regions of the smaller follicles, infact, little more than the end bulb regions, repeatedly stainedpositively with the probes (compare FIGS. 7 and 8), as visualised byeither the digoxygenin or the Spectrum green fluorophore to indicate thecells of male origin. Unfortunately, the morphological resolution of thetissue was not adequate to interpret the probes distribution at thelevel of individual cells, or even tissue layers. Nevertheless, thatboth the fluorophore, (refer to FIG. 9 compared to FIG. 10) anddigoxygenin—(FIG. 7 compared to 8) labelled probe recognised almostidentical regions of the follicles tissue as positive, was considered toreinforce the results.

Experimental Evidence in Support of the Ability of Dermal Sheath Cellsto Provide Long Term Replacement Skin Dermis

Dermal sheath cells were recombined with epidermal cells from hairfollicles and grafted, inside a chamber that separated the graft fromthe surrounding skin cells, onto an animal.

The dermal sheath cells formed a very good dermis with uniform celldensity and no sign of abnormal collagen formation. They also interactedwith the epidermis to produce a thick epidermal covering. A complete andnormal basement membrane was formed between dermal sheath and epidermis.Where the chamber surrounding the graft has been removed, the whiteblock cell infiltrate that has built up outside the graft does notappear to enter the new skin site. Refer to FIG. 11 which represents ahigh power magnification view of the side of a long term [24 days]graft. The line of dark dense white blood cell infiltrate on the left,has not encroached into the graft site. In the dermis, collagen bundlesare structured, dermal cells are regularly distributed and a completeand normal basement membrane is obvious.

Experimental Evidence in Support of Dermal Sheath Cell Stem CellPotential

FIG. 12 (A-I) represent pictorial evidence of dermal sheath cellscapability to differentiate into different mesenchymal cells and hencetheir stem cell potential. It can be seen that these cells candifferentiate into myotubes, adipocytes, chondrocytes and mineralproducing bone cells. Further surprising evidence includes hair follicletissue, obtained from individuals in the 95-105 age range, was found tobe viable and capable of acting as a productive source for cell cultureinitiation. This data supports the hypothesis of the capability of stemcells to differentiate and reproduce remains constant during lifetime(10). Additionally repeated freezing and thawing of primary dermalsheath cells and subsequent cloning did not alter their potential toexhibit at least four different phenotypes despite their prior exposureto adverse conditions.

Experimental Evidence in Support of Dermal Sheath Multipotentiality

Muscle Myotubes

Subpopulations of small spindle-shaped cells were observed bothsingularly and in various states of fusion (as can also be commonly seenin routinely prepared cultures), some forming long branching,multinucleate myotube-like structures. A proportion of these cellsstrained positively with myosin, desmin and/or alpha-smooth muscle actinmonoclonal antibodies. [There have even been an odd occasion in the pastwhen we have observed spontaneous rhythmic beating, i.e. contractions,of long aggregations of such muscle precursor-type cells in our petridishes].

Adipocytes

These cells were identified by their distinctive multivesiculateappearance and the fact that the material contained within theirvesicles was stained red by Sudan IV, and thus shown to be saturatedneutral lipid.

Chondrocytes

Seen as accumulations of rounded cells with pericellular pH 1.0 AlcianBlue positive material which would be chondriotin and keratan sulphateproteoglycans, and lacunae between many of the cells—{interestinglysimilar cell behaviour is observed when rat dermal sheath cells aremixed with microdissected ear cartlidge in vitro}. This also seemslikely to be related to our observations in vivo, when implanted dermalsheath cells appear to stimulate hyperplasia in the normally inactiveear cartilage.

Mineral Producing Bone Cells

These cells were identified by their formation of aggregates in whichthe matrix appeared mineralised and shined positively for calciumphosphates, after being treated by the von Kossa method.

Further distinctive cell types have also been observed in our dermalsheath cell cultures (including interesting dendritic populations) butas yet these remain inaccurately defined.

Experimental Evidence in Support of Dermal Sheath Cells as Substitutesfor Fibroblasts in Skin Wounding

Fluorescent dye (DiI) labelled dermal sheath cells and fibroblasts wereimplanted into skin wounds in a collagen gel, dermal sheath cellssurvived comparably to skin cells over 10 days and were observed topenetrate further into host skin. Dermal sheath cells were also shown tobe capable of migration and incorporating themselves into normal skinaway from the wound itself (refer to FIG. 13 which represents pictorialevidence of skin at the margin of a wound and in which dermal sheathcells have surrounded an isolated follicle remote in undamaged tissue).

Storage of Dermal Sheath Tissue

Our investigations have shown that dermal sheath tissue and/or cellsderived therefrom can be stored long term at low temperatures and yetstill, when subjected to appropriate conditions, grow. This clearly hasimportant implications in the storage of wound healing therapeutics, andspecifically, the storage of grafts or “living skins” made therefrom.

Moreover, our investigations have also shown that the dermal sheathcells can persist for a long time in culture under conditions of extremestress. This has important implications for wound healing therapeuticsderived from this tissue, since it highlights that it is favourablyrobust and also that it displays stem cell characteristic durability andviability.

Evidence in Support of Transfected Dermal Sheath Cells

Using the construct containing enhanced green fluorescent protein e-GFPdepicted in FIG. 14 obtained by the method outlined in FIG. 15. Two setsof dermal sheath cells were transfected on two separate occasions andwere shown to visibly express the GFP by 36 hours (refer to FIG. 16).Ally cells that contained the construct were identified by theirfluorescence. Transfection rates were reasonably high, approaching 20%of the cells. Furthermore, the cells remained green for more than 2weeks. It is our assumption that the cells would survive in vivo if theywere put back into humans/other species either at the same or differentsites.

In short, not only does dermal sheath tissue and/or cells derivedtherefrom and/or cells typically closely associated with hair follicleshave all the advantageous properties that one might hope to find in agene therapy system but they also have properties that facilitate theuse of the tissue and/or cells derived therefrom in terms ofmanufacturing and long term storage.

REFERENCES

-   1. Anderson W. F. (1998). Human gene therapy. Nature 392: 25-30.-   2. Mulligan R. C. (1993). The Basic Science of Gene Therapy. Science    260: 926-932.-   3. Malkinson, F. D. & Keane, J. T. (1978). Hair matrix kinetics: a    selective review. Int J Dermatol. 17, 536-551.-   4. Oliver R. F. & Jahoda C. A. B. (1989). The dermal papilla and    maintenance of hair growth. In The biology of wool and hair.    (ed. G. E. Rogers, P. J. Reis, K. A. Ward, R. C. Marshall),    pp.51-67. Cambridge: Cambridge University Press.-   5. Reynolds, A. J. and Jahoda, C. A. B. (1991a). Inductive    properties of hair follicle cells. In The Molecular and Structural    Biology of Hair. Proc. N.Y. Acad. Sci. 624, 226-242.-   6. Reynolds, A. J. & Jahoda, C. A. B. (1992). Cultured dermal    papilla cells induce follicle formation and hair growth by    transdifferentiation of an adult epidermis. Development 115,    587-593.-   7. Reynolds, A. J., Lawrence, C. & Jahoda, C. A. B. (1993). Culture    of human hair follicle germinative epidermal cells. J. Invest    Dermatol. 101, 634-638.-   8. Oliver, R. F. (1966). Histological studies of whisker    regeneration in the hooded rat. J. Embryol. Exp. Morphol. 16:    231-244.-   9. Jahoda, C. A. B., Horne K. A., Mauger, A., Bard S., & Sengel P.    (1992). Cellular and extracellular involvement in the regeneration    of the rat lower vibrissa follicle. Development 114: 887-897.-   10. Haynesworth, S. E, Goldberg, V. M. & Caplan, A. I. (1993).    Diminution of the number of mesenchymal stem cells as a cause for    skeletal ageing. Chapter 7. In: Musculoskeletal soft-tissue ageing    impact on mobility. [Eds. J. A. Buckwater & V. M. Goldberg]. pp    79-87.

1. A therapeutic composition, comprising isolated hair follicle dermalsheath tissue and/or a cell derived therefrom comprising at least oneselected gene, or functional fragment thereof, and wherein said at leastone selected gene, or functional fragment thereof is delivered to atarget site when said dermal sheath tissue is part of a gene therapyvehicle.
 2. A gene therapy vehicle for delivering at least one selectedgene, or functional fragment thereof, to a target site wherein said genetherapy vehicle comprises isolated hair follicle dermal sheath tissueand/or a cell derived therefrom.
 3. The therapeutic composition of claim1, wherein said dermal sheath tissue or cell is derived from the lowerportion of a hair follicle.
 4. The therapeutic composition of claim 3,wherein said dermal sheath tissue or cell is derived from a lower thirdof said hair follicle.
 5. The therapeutic composition of claim 3,wherein said dermal sheath tissue or said cell is derived from a segmentor ring of a combination of follicle/tissue cells.
 6. The gene therapyvehicle of claim 2, which is engineered by recombinant techniques so asto include at least one insertion site into which at least one selectedgene be placed.
 7. The gene therapy vehicle of claim 6, wherein saidselected gene is inserted into said gene therapy vehicle so that theexpression of said selected gene results in the provision of thecorresponding protein product.
 8. The gene therapy vehicle of claim 7,wherein said vehicle is provided with multiple insertion sites to carrymultiple genes and wherein when said genes are expressed said genetherapy vehicle provides for the delivery of multiple proteins.
 9. Thegene therapy vehicle of claim 2, further comprising a promoter whereinsaid selected gene is under the transcriptional control of saidpromoter.
 10. The gene therapy vehicle of claim 9, wherein said promoteris an inducible promoter.
 11. The gene therapy vehicle of claim 9,wherein said promoter is a constitutive promoter.
 12. A vectorcomprising the gene therapy vehicle of claim 2 wherein said vectorfurther comprises (i) at least one insertion site for at least oneselected gene, or functional fragment thereof, and (ii) other expressioncontrol elements for ensuring that once the vector infects or penetratessaid tissue and/or cells of said gene therapy vehicle, expression ofsaid selected gene can take place.
 13. A therapeutic compositioncomprising a carrier and the gene therapy vehicle of claim
 2. 14. Thetherapeutic composition of claim 13, wherein said composition hasanti-bacterial properties.
 15. The therapeutic composition of claim 13,wherein said composition has anti-septic properties.
 16. The therapeuticcomposition of claim 13, wherein said composition further comprisesgrowth promoting additives.
 17. The therapeutic composition of claim 13,wherein said composition further comprises at least one anaesthetic. 18.The therapeutic composition of claim 13, for topical application whereinsaid therapeutic composition is provided in a suitable carrier solution,gel, cream, or emollient.
 19. The therapeutic composition of claim 13,wherein said therapeutic composition comprises a carrier solution assaid carrier.
 20. A therapeutic appliance comprising the therapeuticcomposition of claim 13, wherein said carrier is incorporated therein,and/or attached thereto, a plaster or bandage.
 21. A gene therapyvehicle for use in delivering a selected gene, or functional fragmentthereof, to a given site wherein said gene therapy vehicle compriseshair follicle dermal sheath tissue and/or a cell derived therefrom,which tissue and/or cells comprise heterologous genetic material andwhich, in vivo, said dermal sheath tissue and/or cell differentiate toprovide at least one differentiated tissue type.
 22. The gene therapyvehicle of claim 21, which acts as a wound healing system.
 23. A woundhealing system comprising a matrix material having incorporated therein,and/or attached thereto, the gene therapy vehicle of claim
 21. 24. Awound healing system according to claim 23 wherein said matrix materialcomprises native collagen.
 25. The wound healing system of claim 23,wherein said matrix material comprises collagenous gels or latticesconstructed from reconstituted collagen.
 26. The wound healing system ofclaim 25, wherein said matrix material comprises components from anextra cellular matrix.
 27. A wound healing system according to claim 26comprising a surgical dressing.
 28. The wound healing system of claim27, for treatment of acute, and/or chronic, and/or minor, and/or severe,wound healing.
 29. A wound healing system according to claim 28 for usein the treatment of cartilage repair, and/or bone repair, and/or musclerepair, and/or connective tissue repair, and/or blood vessel repair. 30.A wound healing system according to claim 29 wherein said systemcomprises a plurality of cell types from a hair follicle.
 31. The woundhealing system of claim 30, wherein one of said cell types, in additionto said dermal sheath tissue, and/or said cell derived therefrom,comprises dermal papilla tissue.
 32. A therapeutic composition accordingto claim 13 wherein said composition comprises a plurality of cell typesfrom a hair follicle.
 33. The therapeutic composition of claim 31,wherein one of said cell types, in addition to said dermal sheathtissue, and/or said cell derived therefrom, comprises dermal papillatissue.